Mb2Cas12a variants with enhanced efficiency

Abstract

The invention provides Cas12a mutants from Murraya bovis AAX08, and methods of using the Cas12a mutants. The Mb2Cas12a mutant comprises a single amino acid substitution, a plurality of amino acid substitutions, a structural domain exchange, or all of the above. Compared with a wild type moraxella bovine eye AAX08Cas12a enzyme, the mutants have enhanced DNA (Deoxyribose Nucleic Acid) lysis activity in plants.

Description

Technical Field

[0001] Described are Cas12a mutants from Moraxella bovoculi AAX08 and methods for their use that have enhanced DNA cleavage activity in plants compared to the wild-type enzyme.

[0002] Sequence Listing

[0003] This application is accompanied by a sequence listing entitled "Mb2Cas12a Variants with EnhancedEfficiency_ST26.xml," created on November 2, 2022, which is approximately 967 kilobytes in size. This sequence listing is incorporated herein by reference in its entirety. This sequence listing is submitted with this application via EFS-Web and complies with 37 CFR §1.824(a)(2)-(6) and (b). Background Art

[0004] Mb2Cas12a from Moraxella bovis AAX08 has been shown to have genome editing capabilities in plants (Zhang et al., 2021), however, its nuclease activity is lower than that of other Cas12a orthologs widely used in plants or mammalian cells, such as LbCas12a, AsCas12a, or FnCas12a (Zetsche et al., 2020; Zhang et al., 2021). Due to its unique properties of high performance at lower temperatures and the potential to recognize shorter PAMs, Mb2Cas12a needs to have improved nuclease activity in plants. Summary of the Invention

[0005] Therefore, wild-type mutant Mb2Cas12a polypeptide variants are needed. The variant may include at least one amino acid substitution introduced into the wild-type Mb2Cas12a polypeptide sequence of SEQ ID NO: 1. The substitution may occur at the following positions: D172, F357, F547, A742, E797, Y819, E913, I914, L917, N918, V921, H939, and / or Y1172. Specific substitutions include: D172R, D172K, F357W, F547Y, A742S, E797A, Y819F, I914K, L917V, N918A, N918K, V921K, V921Q, H939Q, and Y1172N. Specific sequences of desired variants of Mb2Cas12a include SEQ ID NO: 3, 5, 26, 35, 57, 58, 64, 76, 80, 85, 86, 87, 88, or 127.

[0006] Alternative variants can be obtained by swapping a domain from Mb2Cas12a with an orthologous domain from another Cas12a peptide. Domain swapping can occur at the following domains: WED-MR1, WED-MR2, BH, Up-seq, and Nuc. The WED-MR1 domain can be replaced by SEQ ID NO: 24, 29, or 30. The WED-MR2 domain can be replaced by SEQ ID NO: 25, 32, or 33. The BH domain can be replaced by SEQ ID NO: 40, 41, or 42. The Up-seq domain can be replaced by SEQ ID NO: 59, 60, 61, 62, or 63. The Nuc domain can be replaced by SEQ ID NO: 65, 66, and 67. Domain swap variants of Mb2Cas12a can include SEQ ID NO: 37, 59, 60, 61, 62, 63, 74, 75, 77, or 78.

[0007] Mutant Mb2Cas12a polypeptides can also include amino acid substitutions and domain swaps. Examples of such mutants include SEQ ID NO: 36, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, and 107.

[0008] When the mutants described herein are used to contact the plant genome, these mutants are useful in methods for editing plants. As can be seen in SEQ ID NO:18-21, those guide RNAs can also be used in these methods. Practicing these methods will obtain edited plants. Constructs and plasmids encoding these mutant Mb2Cas12a polypeptides can be used to express desired mutants in appropriate organisms and / or tissues. These organisms can be non-human cells, such as plant cells or tissues.

[0009] Brief description of the sequences in the sequence listing

[0010] SEQ ID NO: 1 is the amino acid sequence of wild-type Mb2Cas12a.

[0011] SEQ ID NO: 2 is a nucleotide sequence encoding the amino acid sequence of wild-type Mb2Cas12a.

[0012] SEQ ID NO: 3 is the amino acid sequence of Mb2Cas12a comprising the D172R mutation.

[0013] SEQ ID NO: 4 is a nucleotide sequence encoding the amino acid sequence of Mb2Cas12a comprising the D172R mutation.

[0014] SEQ ID NO: 5 is the amino acid sequence of Mb2Cas12a comprising the D172K mutation.

[0015] SEQ ID NO: 6 is a nucleotide sequence encoding the amino acid sequence of Mb2Cas12a comprising a D172K mutation.

[0016] SEQ ID NO: 7 is the nucleotide sequence of the Sugarcane ubiquitin promoter ("prSoUbi4-02").

[0017] SEQ ID NO: 8 is the amino acid sequence of the SV40 nuclear localization signal ("xSV40NLS-06").

[0018] SEQ ID NO: 9 is the amino acid sequence of a 30 amino acid flexible peptide linker ("x-linker-06") having a repeating motif (GGGGS)6.

[0019] SEQ ID NO: 10 is the amino acid sequence of a short linker of 8 amino acids ("xSGGS linker-02") having a repeating motif (SGGS) 2.

[0020] SEQ ID NO: 11 is the nucleotide sequence of the Agrobacterium tumefaciens nopaline synthase gene terminator ("tNOS-05-01").

[0021] SEQ ID NO: 12 is the nucleotide sequence of a ribozyme ("rHH-05").

[0022] SEQ ID NO: 13 is the nucleotide sequence of the ribozyme ("rHDV-01").

[0023] SEQ ID NO: 14 is the nucleotide sequence of a crRNA array targeting four maize genes ("rMb2gRNACas12aZmWxy1-01, rMb2gRNACas12aZmBX9-A, rMb2gRNACas12aZmGL2-01, rMb2gRNACas12aZmBINa"), including ribozymes.

[0024] SEQ ID NO: 15 is the nucleotide sequence of construct 26411.

[0025] SEQ ID NO: 16 is the nucleotide sequence of construct 26363.

[0026] SEQ ID NO: 17 is the nucleotide sequence of construct 26410.

[0027] SEQ ID NO: 18 is a nucleotide sequence encoding the portion of the guide RNA that hybridizes to the target gene ZmWx1.

[0028] SEQ ID NO: 19 is a nucleotide sequence encoding the portion of the guide RNA that hybridizes to the target gene ZmBx9.

[0029] SEQ ID NO: 20 is a nucleotide sequence encoding the portion of the guide RNA that hybridizes to the target gene ZmGL2.

[0030] SEQ ID NO: 21 is a nucleotide sequence encoding the guide RNA portion that hybridizes to the target gene ZmBINa.

[0031] SEQ ID NO: 22 is the amino acid sequence of WED-MR1-Mb2 (identical to SEQ ID NO: 31).

[0032] SEQ ID NO: 23 is the amino acid sequence of WED-MR2-Mb2 (identical to SEQ ID NO: 32).

[0033] SEQ ID NO: 24 is the amino acid sequence of the WED-MR1 cognate from LbCas12a.

[0034] SEQ ID NO: 25 is the amino acid sequence of the WED-MR2 homolog from LbCas12a.

[0035] SEQ ID NO: 26 is the amino acid sequence of Mb2Cas12a comprising the E797A mutation.

[0036] SEQ ID NO: 27 is the nucleotide sequence of construct 27731.

[0037] SEQ ID NO: 28 is the nucleotide sequence of construct 26840.

[0038] SEQ ID NO: 29 is the amino acid sequence of the WED-MR1 homolog from AsCas12a.

[0039] SEQ ID NO: 30 is the amino acid sequence of the WED-MR1 homolog from FnCas12a.

[0040] SEQ ID NO: 31 is the amino acid sequence of the WED-MR1 homolog from Mb2Cas12a and Mb2Cas12a-22581.

[0041] SEQ ID NO: 32 is the amino acid sequence of the WED-MR2 homolog from AsCas12a.

[0042] SEQ ID NO: 33 is the amino acid sequence of the WED-MR2 homolog from FnCas12a.

[0043] SEQ ID NO: 34 is the amino acid sequence of the WED-MR2 homolog from Mb2Cas12a and Mb2Cas12a-22581.

[0044] SEQ ID NO: 35 is the amino acid sequence of Mb2Cas12a comprising D172R and E797A mutations.

[0045] SEQ ID NO: 36 is the amino acid sequence of Mb2Cas12a comprising D172R and WED-MR1-Lb.

[0046] SEQ ID NO: 37 is the amino acid sequence of Mb2Cas12a comprising WED-MR2-Lb.

[0047] SEQ ID NO: 38 is the nucleotide sequence of construct 26841.

[0048] SEQ ID NO: 39 is the nucleotide sequence of construct 27493.

[0049] SEQ ID NO: 40 is the amino acid sequence of the BH domain of LbCas12a.

[0050] SEQ ID NO: 41 is the amino acid sequence of the BH domain of AsCas12a.

[0051] SEQ ID NO: 42 is the amino acid sequence of the BH domain of FnCas12a.

[0052] SEQ ID NO: 43 is the amino acid sequence of the BH domain of Mb2Cas12a and Mb2Cas12a-22581.

[0053] SEQ ID NO: 44 is the amino acid sequence of the Up-seq region of LbCas12a.

[0054] SEQ ID NO: 45 is the amino acid sequence of the Up-seq region of AsCas12a.

[0055] SEQ ID NO: 46 is the amino acid sequence of the Up-seq region of FnCas12a.

[0056] SEQ ID NO:47 Amino acid sequence of the Up-seq region of Mb2Cas12a-22581.

[0057] SEQ ID NO: 48 is the amino acid sequence of the Up-seq region of Mb2Cas12a.

[0058] SEQ ID NO:49 is the nucleotide sequence of construct 26442.

[0059] SEQ ID NO:50 is the nucleotide sequence of construct 26443.

[0060] SEQ ID NO:51 is the nucleotide sequence of construct 26623.

[0061] SEQ ID NO:52 is the nucleotide sequence of construct 26444.

[0062] SEQ ID NO:53 is the nucleotide sequence of construct 27031.

[0063] SEQ ID NO:54 is the nucleotide sequence of construct 26445.

[0064] SEQ ID NO:55 is the nucleotide sequence of construct 27030.

[0065] SEQ ID NO:56 is the nucleotide sequence of construct 27927.

[0066] SEQ ID NO: 57 is the amino acid sequence of Mb2Cas12a V921Q.

[0067] SEQ ID NO: 58 is the amino acid sequence of Mb2Cas12a V921K.

[0068] SEQ ID NO: 59 is the amino acid sequence of Mb2Cas12a BH1-Lb.

[0069] SEQ ID NO:60 is the amino acid sequence of Mb2Cas12a BH2-Lb.

[0070] SEQ ID NO:61 is the amino acid sequence of Mb2Cas12a BH1-As.

[0071] SEQ ID NO:62 is the amino acid sequence of Mb2Cas12a BH2-As.

[0072] SEQ ID NO: 63 is the amino acid sequence of Mb2Cas12a BH3-As.

[0073] SEQ ID NO: 64 is the amino acid sequence of Mb2Cas12a L917V+N918K.

[0074] SEQ ID NO: 65 is the amino acid sequence of a micro-region in the Nuc domain of LbCas12a.

[0075] SEQ ID NO: 66 is the amino acid sequence of the microregion in the Nuc domain of AsCas12a.

[0076] SEQ ID NO: 67 is the amino acid sequence of the microregion in the Nuc domain of FnCas12a.

[0077] SEQ ID NO: 68 is the amino acid sequence of the microregion in the Nuc domain of Mb2Cas12a and Mb2Cas12a-22581.

[0078] SEQ ID NO:69 is the nucleotide sequence of construct 26440.

[0079] SEQ ID NO:70 is the nucleotide sequence of construct 26441.

[0080] SEQ ID NO:71 is the nucleotide sequence of construct 26438.

[0081] SEQ ID NO:72 is the nucleotide sequence of construct 26553.

[0082] SEQ ID NO:73 is the nucleotide sequence of construct 27215.

[0083] SEQ ID NO:74 is the amino acid sequence of Mb2Cas12a Nuc1-Lb.

[0084] SEQ ID NO:75 is the amino acid sequence of Mb2Cas12a Nuc1-As.

[0085] SEQ ID NO: 76 is the amino acid sequence of Mb2Cas12a Y1172N.

[0086] SEQ ID NO:77 is the amino acid sequence of Mb2Cas12a Nuc2-Lb.

[0087] SEQ ID NO:78 is the amino acid sequence of Mb2Cas12a Nuc2-As.

[0088] SEQ ID NO:79 is the nucleotide sequence of construct 26446.

[0089] SEQ ID NO: 80 is the amino acid sequence of Mb2Cas12a F357W.

[0090] SEQ ID NO:81 is the nucleotide sequence of construct 27495.

[0091] SEQ ID NO:82 is the nucleotide sequence of construct 27745.

[0092] SEQ ID NO:83 is the nucleotide sequence of construct 27747.

[0093] SEQ ID NO:84 is the nucleotide sequence of construct 27501.

[0094] SEQ ID NO: 85 is the amino acid sequence of Mb2Cas12a F547Y.

[0095] SEQ ID NO: 86 is the amino acid sequence of Mb2Cas12a A742S.

[0096] SEQ ID NO: 87 is the amino acid sequence of Mb2Cas12a Y819F.

[0097] SEQ ID NO: 88 is the amino acid sequence of Mb2Cas12a H939Q.

[0098] SEQ ID NO: 89 is the amino acid sequence of Mb2Cas12a BH2-As+D172R.

[0099] SEQ ID NO:90 is the amino acid sequence of Mb2Cas12a BH2-As+Nuc1-As+F357W.

[0100] SEQ ID NO:91 is the amino acid sequence of Mb2Cas12a BH2-As+Nuc1-As+F357W+D172R.

[0101] SEQ ID NO:92 is the amino acid sequence of Mb2Cas12a BH2-As+Nuc1-As+F357W+V921K.

[0102] SEQ ID NO:93 is the amino acid sequence of Mb2Cas12a BH2-As+Nuc1-As+F357W+V921K+D172R.

[0103] SEQ ID NO:94 is the amino acid sequence of Mb2Cas12a BH2-As+Nuc1-As+F357W+V921K+E797A.

[0104] SEQ ID NO:95 is the amino acid sequence of Mb2Cas12a BH2-As+Nuc1-As+F357W+V921K+E797A+D172R.

[0105] SEQ ID NO:96 is the amino acid sequence of Mb2Cas12a BH2-As+Nuc1-As+F357W+V921K+E797A+WED-MR1.

[0106] SEQ ID NO: 97 is the amino acid sequence of Mb2Cas12a BH2-As+Nuc1-As+F357W+V921K+E797A+WED-MR1+D172R.

[0107] SEQ ID NO:98 is the amino acid sequence of Mb2Cas12a BH2-As+E797A.

[0108] SEQ ID NO:99 is the amino acid sequence of Mb2Cas12a BH2-As+E797A+D172R.

[0109] SEQ ID NO: 100 is the amino acid sequence of Mb2Cas12a BH2-As+E797A+F357W.

[0110] SEQ ID NO: 101 is the amino acid sequence of Mb2Cas12a BH2-As+E797A+F357W+D172R.

[0111] SEQ ID NO: 102 is the amino acid sequence of Mb2Cas12a V921K+E797A.

[0112] SEQ ID NO: 103 is the amino acid sequence of Mb2Cas12a V921K+E797A+D172R.

[0113] SEQ ID NO: 104 is the amino acid sequence of Mb2Cas12a V921K+E797A+F357W.

[0114] SEQ ID NO: 105 is the amino acid sequence of Mb2Cas12a V921K+E797A+F357W+D172R.

[0115] SEQ ID NO: 106 is the amino acid sequence of Mb2Cas12a BH2-As+Nuc1-As+F357W+E797A.

[0116] SEQ ID NO: 107 is the amino acid sequence of Mb2Cas12a BH2-As+Nuc1-As+F357W+E797A+D172R.

[0117] SEQ ID NO: 108 is the nucleotide sequence of construct 27218.

[0118] SEQ ID NO: 109 is the nucleotide sequence of construct 27025.

[0119] SEQ ID NO: 110 is the nucleotide sequence of construct 27216.

[0120] SEQ ID NO: 111 is the nucleotide sequence of construct 27219.

[0121] SEQ ID NO: 112 is the nucleotide sequence of construct 27220.

[0122] SEQ ID NO: 113 is the nucleotide sequence of construct 27225.

[0123] SEQ ID NO: 114 is the nucleotide sequence of construct 27228.

[0124] SEQ ID NO: 115 is the nucleotide sequence of construct 27223.

[0125] SEQ ID NO: 116 is the nucleotide sequence of construct 27224.

[0126] SEQ ID NO: 117 is the nucleotide sequence of construct 27310.

[0127] SEQ ID NO: 118 is the nucleotide sequence of construct 27311.

[0128] SEQ ID NO: 119 is the nucleotide sequence of construct 27320.

[0129] SEQ ID NO: 120 is the nucleotide sequence of construct 27321.

[0130] SEQ ID NO: 121 is the nucleotide sequence of construct 27322.

[0131] SEQ ID NO: 122 is the nucleotide sequence of construct 27381.

[0132] SEQ ID NO: 123 is the nucleotide sequence of construct 27382.

[0133] SEQ ID NO: 124 is the nucleotide sequence of construct 27383.

[0134] SEQ ID NO: 125 is the nucleotide sequence of construct 27325.

[0135] SEQ ID NO: 126 is the nucleotide sequence of construct 27323.

[0136] SEQ ID NO: 127 is the amino acid sequence of Mb2Cas12a I914K+L917V+N918A.

[0137] SEQ ID NO: 128 is the nucleotide sequence of construct 27926. BRIEF DESCRIPTION OF THE DRAWINGS

[0138] Figure 1 The general configuration of the expression cassette is shown. This same configuration was used for each Mb2Cas12a variant tested.

[0139] definition

[0140] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by those of ordinary skill in the art. References to the techniques employed herein are intended to refer to techniques commonly understood in the art, including modifications of those techniques and / or equivalent technical alternatives that are clear to those of ordinary skill in the art. Although it is believed that the following terms may be well understood by those of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the subject matter disclosed herein.

[0141] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an enzyme" optionally includes combinations of two or more such molecules, etc.

[0142] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0143] As used herein, the term "about" refers to the usual error range for the corresponding value that is readily known to those skilled in the art, for example, ±20%, ±10% or ±5% within the intended meaning of the recited value.

[0144] As used herein, the term "comprising" or "comprises" is open ended. When used in conjunction with a subject nucleic acid (or amino acid sequence), it refers to a nucleic acid sequence (or amino acid sequence) that includes the subject sequence as a portion or as its entire sequence.

[0145] As used herein, the transition phrase "consisting essentially of" means that the scope of a claim is interpreted to encompass the specified materials or steps recited in the claim, as well as those materials or steps that do not materially affect one or more of the basic and novel characteristics of the claimed subject matter. Therefore, when used in the claims of the present disclosure, the term "consisting essentially of" is not intended to be interpreted as equivalent to "comprising."

[0146] The term "plurality" refers to more than one entity. Thus, a "majority of individuals" refers to at least two individuals. In some embodiments, the term "majority" refers to more than half of a whole. For example, in some embodiments, a "majority of a group" refers to more than half of the members of that group.

[0147] As used herein, term " plant " refers to any plant, particularly seed plant in any developmental stage. As used herein, term " plant cell " refers to the structure and physiological unit of plant, including protoplast and cell wall. Plant cell can be in the form of isolated single cell or cultured cell, or as a part of higher organization unit (such as, plant tissue, plant organ or whole plant). Plant cell can derive from angiosperms or gymnosperms or be a part of them. Plant cell can be monocotyledonous plant cell (for example, maize cell, rice cell, sorghum cell, sugarcane cell, barley cell, wheat cell, oat cell, lawn grass cell or ornamental grass cell) or dicotyledonous plant cell (for example, tobacco cell, pepper cell, eggplant cell, sunflower cell, cruciferous plant cell, flax cell, potato cell, cotton cell, soybean cell, sugar beet cell or rape cell). As used herein, the term "plant cell culture" means a culture of plant units (such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes, and embryos at different developmental stages). As used herein, the term "plant tissue" refers to a group of plant cells organized into structural and functional units. Includes any plant tissue in a plant or in culture. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures, and any plant cell groups organized into structural and / or functional units. The term is not intended to exclude any other type of plant tissue by combining or applying alone with any specific type of plant tissue as listed above or otherwise encompassed by this definition. As used herein, the term "plant part" refers to a part of a plant, including single cells and cell tissues (such as complete plant cells in a plant), cell clumps, and tissue cultures that can regenerate plants. Examples of plant parts include, but are not limited to, single cells and tissues from: pollen, ovules, zygotes, leaves, embryos, roots, root tips, anthers, flowers, floral parts, fruits, stems, buds, cuttings, and seeds; as well as pollen, ovules, egg cells, zygotes, leaves, embryos, roots, root tips, anthers, flowers, floral parts, fruits, stems, buds, cuttings, scions, rhizomes, seeds, protoplasts, callus, and the like.

[0148] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, these terms encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0149] The terms "nucleic acid" and "polynucleotide" are used interchangeably and, as used herein, refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form, and polymers thereof, and refer to both the sense and antisense strands of RNA, cDNA, genomic DNA, mitochondrial DNA, and synthetic forms and mixed polymers thereof. In higher plants, DNA is the genetic material, while RNA is involved in the transfer of information contained within DNA into proteins. A "genome" is the entirety of the genetic material contained in each cell of an organism. It should be understood that when RNA is described, its corresponding cDNA is also described, with uridine represented as thymidine. In specific embodiments, a nucleotide refers to a ribonucleotide, a deoxynucleotide, or a modified form of any type of nucleotide, and combinations thereof. In addition, the polynucleotides disclosed herein may include either or both naturally occurring nucleotides and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide linkages. Nucleic acid molecules may be chemically or biochemically modified, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Such modification includes, for example, tags, methylations, substitutions of one or more naturally occurring nucleotides, internucleotide modifications such as uncharged linkages (for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (for example, phosphorothioates, phosphorodithioates, etc.), side groups (for example, polypeptides), intercalators (for example, acridine, psoralens, etc.), chelating agents, alkylating agents, and modified linkages (for example, α-anomeric nucleic acids, etc.). The above terms are also intended to include any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hairpin-shaped, circular, and padlock-shaped conformations. Unless otherwise indicated, reference to a nucleic acid sequence encompasses its complementary sequence. Therefore, reference to a nucleic acid molecule with a specific sequence is understood to encompass its complementary chain with its complementary sequence. When nucleotide sequences specifically hybridize in solution, these nucleotide sequences are "complementary" (for example, according to Watson-Crick base pairing principles). The term also includes codon-optimized nucleic acids encoding identical polypeptide sequences. It is also understood that nucleic acids can be unpurified, purified, or attached to, for example, a synthetic material such as a bead or column matrix.

[0150] In the context of nucleic acid sequences, the term "corresponding to" means that when the nucleic acid sequences of certain sequences are aligned with each other, the nucleic acids "corresponding to" certain enumerated positions in the present invention are those aligned with these positions in the reference sequence, but are not necessarily located in these precise numerical positions relative to the specific nucleic acid sequence of the present invention. Optimal alignment of sequences for comparison can be performed by computerized implementations of known algorithms or by visual inspection. Easily available sequence comparison and multiple sequence alignment algorithms are the Basic Local Alignment Search Tool (BLAST) and ClustalW / ClustalW2 / Clustal Omega programs available on the Internet (e.g., the website of EMBL-EBI), respectively. Other suitable programs include, but are not limited to, GAP, BestFit, Plot Similarity, and FASTA, which are part of the Accelrys GCG software package available from Accelrys Corporation (San Diego, California, USA). See also Smith and Waterman, 1981; Needleman and Wunsch, 1970; Pearson and Lipman, 1988; Ausubel et al., 1988; and Sambrook and Russell, 2001.

[0151] Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly specified. In particular, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues. See Batzer et al., Nucleic Acids Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994).

[0152] As used in the context of polynucleotide or polypeptide sequences described herein, the terms "identity" or "substantial identity" refer to sequences that have at least 60% sequence identity to a reference sequence. Alternatively, the percent identity can be any integer from 60% to 100%. Exemplary embodiments include at least: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% as compared to a reference sequence, as used using the programs described herein, preferably BLAST using standard parameters as described below. One skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, etc.

[0153] For sequence comparison, typically, a kind of sequence serves as the reference sequence that is compared with the test sequence.When using a sequence comparison algorithm, test sequence and reference sequence are input into a computer, and subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. Then, the sequence comparison algorithm will calculate the sequence identity percentage of the test sequence relative to the reference sequence based on the program parameters.

[0154] As used herein, "comparison window" includes reference to a segment having any one of the number of consecutive positions selected from the group consisting of from 20 to 600, typically about 50 to about 200, more typically about 100 to about 150, wherein a sequence can be compared to a reference sequence having the same number of consecutive positions after the two sequences are optimally aligned. Methods of sequence alignment for comparison are well known in the art. Optimal alignment of sequences for comparison can be achieved by the local homology algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981); the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970); the search by similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. 85:2444 (1988); computer implementations of these algorithms (e.g., BLAST); or by manual alignment and visual inspection.

[0155] A "gene" is a defined region within a genome that, in addition to the aforementioned coding nucleic acid sequence, also contains other major regulatory nucleic acid sequences responsible for controlling the expression (i.e., transcription and translation) of the coding portion. A gene can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences, and 5' and 3' untranslated regions). A gene typically expresses mRNA, functional RNA, or a specific protein, including regulatory sequences. A gene may or may not be used to produce a functional protein. In some embodiments, a gene refers only to the coding region. The term "natural gene" refers to a gene as found in nature. The term "chimeric gene" refers to any gene that contains: 1) a DNA sequence including regulatory and coding sequences that are not found together in nature, or 2) a sequence encoding a portion of a protein that is not naturally adjacent, or 3) a portion of a promoter that is not naturally adjacent. Thus, a chimeric gene can contain regulatory and coding sequences obtained from different sources, or regulatory and coding sequences obtained from the same source but arranged in a manner different from that found in nature. A gene can be "isolated," which means a nucleic acid molecule that is substantially or essentially free from components normally associated with the nucleic acid molecule in its natural state. Such components include other cellular material, culture medium from recombinant production, and / or various chemicals used in the chemical synthesis of nucleic acid molecules.

[0156] "Isolated" nucleic acid molecules or nucleotide sequence or "isolated" polypeptide are nucleic acid molecules, nucleotide sequence or polypeptides that exist by artificially breaking away from their natural environment and / or have different, modified, regulated and / or changed functions and therefore are not natural products.Isolated nucleic acid molecules or isolated polypeptides can exist in purified form or can be present in non-natural environments (such as, for example, recombinant host cells).Therefore, for example, with respect to polynucleotides, the meaning of the term isolated is to separate polynucleotides from their naturally occurring chromosomes and / or cells therein. If a kind of polynucleotides is separated from its naturally occurring chromosomes and / or cells therein, and then inserted into genetic background, chromosomes, chromosomal position and / or cells therein that are not naturally present in, then the polynucleotides are also isolated.Recombinant nucleic acid molecules and nucleotide sequence of the present invention can be considered as "isolated" as defined above.

[0157] Thus, an "isolated nucleic acid molecule" or "isolated nucleotide sequence" is a nucleic acid molecule or nucleotide sequence that is not immediately adjacent to the nucleotide sequences immediately adjacent to it (the sequences at the 5' end and the sequences at the 3' end) in the naturally occurring genome of the organism from which it is derived. Thus, in one embodiment, an isolated nucleic acid includes some or all of the 5' non-coding (e.g., promoter) sequences that are immediately adjacent to the coding sequence. Thus, the term includes, for example, recombinant nucleic acids that are incorporated into a vector, into a self-replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or that exist as a separate molecule (e.g., a cDNA or genomic DNA fragment generated by PCR or restriction endonuclease treatment) independent of other sequences. It also includes recombinant nucleic acids that are part of a hybrid nucleic acid molecule that encodes another polypeptide or peptide sequence. An "isolated nucleic acid molecule" or "isolated nucleotide sequence" may also include a nucleotide sequence that is derived from and inserted into the same natural original cell type, but is present in a non-natural state, for example, in a different copy number, and / or under the control of regulatory sequences that are different from those found in the natural state of the nucleic acid molecule.

[0158] The term "isolated" may further refer to a nucleic acid molecule, nucleotide sequence, polypeptide, peptide or fragment that is substantially free of cellular material, viral material and / or culture medium (e.g., when produced by recombinant DNA techniques), or chemical precursors or other chemicals (e.g., when chemically synthesized). Additionally, an "isolated fragment" is a fragment of a nucleic acid molecule, nucleotide sequence or polypeptide that does not naturally occur as a fragment and would not so exist in nature. "Isolated" does not necessarily mean that the preparation is industrially pure (homogeneous), but that it is sufficiently pure to provide the polypeptide or nucleic acid in a form that can be used for its intended purpose.

[0159] "Homology-dependent repair" or "homology-directed repair" or "HDR" refers to a mechanism for repairing ssDNA and double-stranded DNA (dsDNA) damage in cells. This repair mechanism can be utilized by cells in the presence of an HDR template with a sequence that has significant homology to the site of damage. The term "perfect HDR" refers to a situation in which the genomic homology junctions in the replaced allele undergo complete HDR, and "imperfect HDR" refers to a situation in which the genomic homology junctions in the replaced allele undergo partial or incomplete HDR. A donor DNA molecule with homology to the cleavage target DNA sequence is used as a template for repairing the cleavage target DNA sequence, thereby generating a transfer of genetic information from the donor polynucleotide to the target DNA. In this way, new nucleic acid material can be inserted / copied into the site. In some cases, the target DNA is contacted with a donor molecule (e.g., a donor DNA molecule). In some cases, the donor DNA molecule is introduced into the cell. In some cases, at least one segment of the donor DNA molecule is integrated into the genome of the cell.

[0160] "Microhomology-mediated end joining" or "MMEJ" or "alternative non-homologous end joining" (Alt-NHEJ) refers to a form of repair of double-strand breaks in DNA. This repair mechanism utilizes microhomology sequences to align the broken strands. "Non-homologous end joining" or "NHEJ" refers to a form of repair of double-strand breaks in DNA. Double-strand breaks are repaired by direct ligation of the broken ends to each other. Generally, no new nucleic acid material is inserted into the site, but some nucleic acid material may be lost or added, resulting in small deletions or insertions.

[0161] The proteins provided herein include site-directed polypeptides. A "site-directed modifying polypeptide" modifies a target DNA (e.g., via cleavage or methylation of the target DNA) and / or a polypeptide associated with the target DNA (e.g., methylation or acetylation of a histone tail). In some embodiments, due to the association of the site-directed modifying polypeptide with the guide RNA, the site-directed modifying polypeptide interacts with the guide RNA (the guide RNA is a single RNA molecule or an RNA duplex of at least two RNA molecules) and is guided to a DNA sequence (e.g., a chromosomal sequence or an extrachromosomal sequence, such as an episomal sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, etc.). In some embodiments, the site-directed polypeptide is a site-directed nuclease that is capable of cleaving one or both chains of DNA at a specified target sequence.

[0162] The term "cleavage" or "cleaving" refers to the fracture of the covalent phosphodiester linkage in the ribosylphosphodiester backbone of a polynucleotide, and encompasses both single-strand breaks and double-strand breaks. As a result of two different single-strand cleavage events, double-strand cleavage can occur. Cracks can result in blunt ends or staggered ends (also referred to as sticky ends). A "nuclease cleavage site" or "genomic nuclease cleavage site" is a region of nucleotides where a site-directed nuclease cracks (e.g., when bound to a proximal binding site). When a polynucleotide is DNA (e.g., genomic DNA), one or both chains can be cracked at the nuclease cleavage site. This cracking performed by nucleases triggers a DNA repair mechanism in the cell, which establishes an environment where homologous recombination occurs.

[0163] The site-directed nuclease can be a naturally occurring site-directed nuclease. Exemplary naturally occurring site-directed nucleases are known in the art (see, for example, Makarova et al., 2017, Cell 168:328-328.e1 and Shmakov et al., 2017, Nat Rev Microbiol 15(3):169-182, both of which are incorporated herein by reference). In some embodiments, the site-directed nuclease binds to a DNA-targeting polynucleotide (e.g., a guide RNA) and is thereby directed to a specific sequence within the target DNA and cleaves the target DNA.

[0164] In some embodiments, the site-directed nuclease is modified relative to its native sequence (e.g., via mutation of one or more amino acid residues) to alter its function. For example, the site-directed nuclease can be modified to be enzymatically inactive. The term "enzymatically inactive" can mean that the site-directed nuclease can bind to a nucleic acid sequence in a polynucleotide in a sequence-specific manner, but cannot cleave the target polynucleotide. An enzymatically inactive site-directed polypeptide can comprise an enzymatically inactive domain (e.g., a nuclease domain). Enzymatically inactive can mean inactive. Enzymatically inactive can mean substantially inactive. Enzymatically inactive can mean essentially inactive. Enzymatically inactive can mean an activity that is no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 6%, no more than 7%, no more than 8%, no more than 9%, or no more than 10% active compared to an exemplary wild-type activity.

[0165] In some embodiments, the site-directed nuclease comprises a CRISPR-associated (Cas) protein or a Cas nuclease that functions in a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas system. In bacteria, this system can provide adaptive immunity against foreign DNA (Barrangou, R. et al., “CRISPR provides acquired resistance against viruses in prokaryotes,” Science (2007) 315:1709-1712; Makarova, KS et al., “Evolution and classification of the CRISPR-Cas systems,” Nat Rev Microbiol (2011) 9:467-477; Garneau, JE et al., “The CRISPR / Cas bacterial immune system cleaves bacteriophage and plasmid DNA,” Nature (2010) 468:67-71; Sapranauskas, R. et al., “The Streptococcus thermophilus CRISPR / Cas system provides immunity in Escherichia coli,” Nucleic Acids Res (2011) 39:9275-9282). CRISPR / Cas systems (e.g., modified and / or unmodified) can be used as genome engineering tools in a wide variety of organisms, including different mammals, animals, plants, microorganisms, and yeast. The CRISPR / Cas system can include a guide nucleic acid, such as a guide RNA (gRNA), complexed with a Cas protein for targeted regulation of gene expression and / or activity or nucleic acid editing. The RNA-guided Cas protein (e.g., a Cas nuclease such as the Cas9 nuclease) can specifically bind to a target polynucleotide (e.g., DNA) in a sequence-dependent manner.Cas proteins can cleave DNA if they have nuclease activity (Gasiunas, G. et al., “Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria,” Proc Natl Acad Sci USA (2012) 109: E2579-E286; Jinek, M. et al. “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science (2012) 337: 816-821; Sternberg, SH et al. “DNA interrogation by the CRISPR RNA-guided endonuclease Cas9 [CRISPR

[0014] "DNA interrogation with the RNA-guided endonuclease Cas9," Nature (2014) 507:62; Deltcheva, E. et al., "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III," Nature (2011) 471:602-607). DNA cleavage (e.g., double-strand break) can generate DNA break repair, thereby allowing the introduction of one or more genetic modifications (e.g., nucleic acid editing). DNA break repair can occur via non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), or homology-directed repair (HDR). In some embodiments, donor nucleic acids are used to promote HDR, as described in detail below in the "Systems" section.CRISPR-Cas systems have been widely used for programmable genome editing in a variety of organisms and model systems (Cong, L. et al. “Multiplex genome engineering using CRISPR Cas systems,” Science (2013) 339:819-823; Jiang, W. et al. “RNA-guided editing of bacterial genomes using CRISPR-Cas systems,” Nat. Biotechnol. (2013) 31:233-239; Sander, JD and Joung, JK, “CRISPR-Cas systems for editing, regulating and targeting genomes,” Nature Biotechnol. (2014) 32:347-355).

[0166] In some embodiments, the site-directed nuclease described herein includes a Cas protein (further described below in the "system" section) that forms a complex with a guide nucleic acid (such as a guide RNA). In some embodiments, the site-directed nuclease includes a Cas protein that forms a complex with a single guide nucleic acid (such as a single guide RNA (sgRNA)). In some embodiments, the site-directed nuclease includes an RNA binding protein (RBP) that is optionally compounded with a guide nucleic acid (such as a guide RNA (e.g., sgRNA)), which is capable of forming a complex with the Cas protein. In some cases, the RNA-guided Cas protein recognizes a DNA target that is complementary to a portion of a gRNA (referred to as a CRISPRRNA (crRNA) sequence). The target sequence is often referred to as a protospacer, and the crRNA sequence portion that is complementary to the protospacer is often referred to as a spacer. In order to work (e.g., to crack DNA), many Cas nucleases also require a specific protospacer adjacent motif (PAM) (a kind of approximately 2 to 6 base pair DNA sequence) immediately following the protospacer sequence.

[0167] Cas proteins from various species (e.g., those disclosed in Shmakov et al., 2017, or polypeptides derived therefrom) may require different PAM sequences in the target DNA. Therefore, for a particular Cas enzyme selected, the PAM sequence requirement may be different from the 5'-NGG-3' sequence (where N is A, T, C, or G) known to be required for Cas9 activity. Many Cas9 orthologs from a variety of species have been identified, and these proteins share only a few identical amino acids. All identified Cas9 orthologs have a domain architecture identical to the central HNH endonuclease domain and the split-type RuvC / RNase H domain. Cas9 proteins share four key motifs with a conserved architecture; motifs 1, 2, and 4 are RuvC-like motifs, while motif 3 is an HNH motif. In contrast, Cas12a proteins from different species may have different PAM sequence requirements compared to the LbCas12a canonical PAM of TTTV.

[0168] Any suitable CRISPR / Cas system can be used. A variety of nomenclature systems can be used to refer to the CRISPR / Cas system. Exemplary nomenclature systems are provided in Makarova, KS et al., "An updated evolutionary classification of CRISPR-Cas systems," Nat Rev Microbiol (2015) 13: 722-736 and Shmakov, S. et al., "Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems," Mol Cell (2015) 60: 1-13. The CRISPR / Cas system can be a type I, type II, type III, type IV, type V, type VI system or any other suitable CRISPR / Cas system. As used herein, the CRISPR / Cas system can be a class 1, class 2 or any other suitable classification CRISPR / Cas system. Class 1 or class 2 determination can be based on the gene encoding the effector module. Class 1 systems generally have multi-subunit crRNA-effector complexes, while Class 2 systems generally have single proteins, such as Cas9, Cpf1, C2c1, C2c2, C2c3 or crRNA-effector complexes. Class 1 CRISPR / Cas systems can use the complex of multiple Cas proteins to realize regulation and control. Class 1 CRISPR / Cas systems can include, for example, type I (e.g., I, IA, IB, IC, ID, IE, IF, IU), type III (e.g., III, IIIA, IIIB, IIIC, IIID) and type IV (e.g., IV, IVA, IVB) CRISPR / Cas types. Class 2 CRISPR / Cas systems can use single large Cas proteins to realize regulation and control. Class 2 CRISPR / Cas systems can include, for example, type II (e.g., II, IIA, IIB) and type V CRISPR / Cas types. CRISPR systems can complement each other, and / or can promote CRISPR locus targeting by means of trans functional units.

[0169] The Cas protein can be from any suitable organism. Non-limiting examples include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinae spiralis, Streptomyces viridochromo genes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibirica, Streptococcus rapa, Streptococcus spp., Streptococcus pyogenes, Streptococcus spp., Streptococcus spp., Streptococcus spp., Streptococcus spp., Streptococcus spp., Streptococcus spp., Streptococcus spp. sibiricum), Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthhalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Pseudomonas aeruginosa, Synechococcus sp.), Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus varroa watsoni), Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosiphoafricanus, Acaryochloris marina), Leptotrichia shahii, and Francisella novicida. In some aspects, the organism is Streptococcus pyogenes (S.pyogenes). In some aspects, the organism is Staphylococcus aureus (S. aureus). In some aspects, the organism is Streptococcus thermophilus (S. thermophilus).

[0170] Cas proteins can be derived from a variety of bacterial species, including but not limited to Veillonella atypical, Fusobacterium nucleatum, Filifactor alocis, Solobacterium moorei, Coprococcus catus, Treponema denticola, Peptoniphilus duerdenii, Catenibacterium mitsuokai, Streptococcus mutans, Listeria innocua, Staphylococcus pseudintermedius, Acidaminococcus intestine, Olsenella uli, Oenococcus kitaharae, Bifidobacterium bifidum. bifidum), Lactobacillus rhamnosus, Lactobacillus gasseri, Fingoldia grandiflora, Mycoplasma mobile, Mycoplasma gallisepticum, Mycoplasma ovipneumoniae, Mycoplasma canis, Mycoplasma synoviae, Eubacterium rectale, Streptococcus thermophilus, Eubacterium dolichum, Lactobacillus coryniformis subsp.Torquens), Ilyobacter polytropus, Ruminococcus albus, Akkermansia muciniphila, Acidothermus cellulolyticus, Bifidobacterium longum, Bifidobacterium dentium, Corynebacterium diphtheria, Elusimicrobium minutum, Nitratifractorsalsuginis, Sphaerochaeta globus, Fibrobacter succinogenes subsp. Succinogenes, Bacteroides fragilis, Capnocytophaga ochracea, Rhodopseudomonas palustris palustris), Prevotella micans, Prevotella ruminicola, Flavobacterium columnare, Aminomonas paucivorans, Rhodospirillum rubrum, Candidatus Puniceispirillum marinum, Verminephrobactereiseniae, Ralstonia syzygii, Dinoroseobacter shibae, Azospirillum, Nitrobacter hamburgensis, Bradyrhizobium, Wolinella succinogenes, Campylobacter jejuni subsp.Jejuni), Helicobacter mustelae, Bacillus cereus, Acidovorax ebreus, Clostridium perfringens, Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria meningitidis, Pasteurella multocida subsp. Multocida, Sutterella wadsworthensis, Proteobacterium, Legionella pneumophila, Parasutterella excrementihominis, Wolinella succinogenes, and Francisella novicida.

[0171] Non-limiting examples of Cas proteins include c2c1, C2c2, c2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas, Cas6e, Cas6f, Cas7, Cas8a, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Cpf1, Csy1, Csy2, Csy3, Csel (CasA) , Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csxl, Csx15, Csfl, Csf2, Csf3, Csf4 and Cul966, and homologs or modified forms thereof. In some embodiments, the site-directed nuclease of the fusion protein provided herein includes a CRISPR-associated nuclease, wherein the CRISPR-associated nuclease is Cas5, Cas6, Cas7, Cas8, Cas9, Cas12a, Cas12b, Cas12i, Cas12j, Cas12L, Cas12e, Cas12c, Cas12d, Cas12g, Cas12h, TnpB, Cas13a, Cas13b or Cas14. In some embodiments, the CRISPR-associated nuclease is a Cas9 enzyme. In some embodiments, the CRISPR-associated nuclease is a Cas12a enzyme. In some embodiments, the CRISPR-associated nuclease is a nickase or an inactivated form of a CRISPR-associated nuclease.

[0172] Lachnospiraceae bacterium Cpf1 (LbCpf1) is one of a large group of many Cpf1 proteins. The terms "Cpf1" and "Cas12a" are used interchangeably throughout this disclosure. Cpf1 is a Cas protein. In some embodiments, the site-directed nuclease is a catalytically inactive Cas12a from Lachnospiraceae bacteria ("dLbCas12a"). In other embodiments, the site-directed nuclease is a catalytically active Cas12a from Lachnospiraceae bacteria ("LbCas12a") or Moraxella bovis AAX08_00205 ("Mb2Cas12a"). In some embodiments, the site-directed nuclease domain of the fusion protein is a Cas12a protein from any one of the following: Lachnospiraceae, Acidaminococcus sp., Moraxella bovis, Thiomicrospira sp., Moraxella lacunata, Methanomethylophilus alvus, Btyrivibrio sp., or Bacteroidetesoral sp.

[0173] As used herein, the term "domain" refers to a discrete, independently folded unit of amino acid residues. The size of a domain varies, depending on its function and the source organism, from about 25-30 amino acid residues to about 300 residues - the average size of a domain is about 100 residues. Large proteins typically contain more than one domain. A domain can be composed of a combination of motifs. In globular proteins, a motif can be understood as a segment of one or more α-helices and / or one or more β-strands connected by loops to form a repeating pattern. Generally, see Principles of Biochemistry (2nd ed.), 92-96.

[0174] The Cas protein may comprise one or more domains. Non-limiting examples of domains include guiding nucleic acid recognition and / or binding domains, nuclease domains (e.g., DNA enzyme or RNA enzyme domains, RuvC, HNH), DNA binding domains, RNA binding domains, helicase domains, protein-protein interaction domains, and dimerization domains. Guiding nucleic acid recognition and / or binding domains may interact with guiding nucleic acids. The nuclease domain may include catalytic activity for nucleic acid cracking. The nuclease domain may lack catalytic activity to prevent nucleic acid cracking. The Cas protein may be a chimeric Cas protein fused with other proteins or polypeptides. The Cas protein may be a chimera of various Cas proteins, such as comprising domains from different Cas proteins.

[0175] As used herein, "domain swapping" refers to the replacement of a domain by another domain. The replaced domain can be an identified domain, a hypothetical domain, a microregion, or a motif. As used herein, "microregion" refers to the region of at least two amino acid residues within a domain. By way of example (but not limitation), the domain that can be domain swapped includes WED-MR1, WED-MR2, BH, Up-seq, or Nuc or a combination thereof. Further by way of example, those skilled in the art can clone the LbCas12a WED-MR1 coding domain into the nucleotide sequence encoding Mb2Cas12a, thereby replacing the corresponding Mb2Cas12a WED-MR1 domain.

[0176] As used herein, the Cas protein can be an active variant, an inactive variant or a fragment of a wild-type or modified Cas protein. Relative to the wild-type form of the Cas protein, the Cas protein can include amino acid changes, such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras or any combination thereof. The Cas protein can be a polypeptide having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity or sequence similarity with a wild-type exemplary Cas protein. The Cas protein can be a polypeptide having at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% sequence identity and / or sequence similarity with a wild-type exemplary Cas protein. The variant or fragment can comprise at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity or sequence similarity to a wild-type or modified Cas protein or portion thereof. The variant or fragment can be targeted to a nucleic acid locus complexed with a guide nucleic acid while lacking nucleic acid cleavage activity.

[0177] The Cas protein can be modified to optimize the regulation of gene expression. The Cas protein can be modified to increase or decrease nucleic acid binding affinity, nucleic acid binding specificity and / or enzymatic activity. The Cas protein can also be modified to change any other activity or property of the protein, such as stability. For example, one or more nuclease domains of the Cas protein can be modified, deleted or inactivated, or the Cas protein can be truncated to remove domains that are unnecessary for protein function or to optimize (e.g., enhance or reduce) the activity of the Cas protein in order to regulate gene expression.

[0178] One or more nuclease domains (e.g., RuvC, HNH) of Cas protein can be deleted or mutated so that they are no longer functional or contain reduced nuclease activity. For example, in a Cas protein (e.g., Cas9) comprising at least two nuclease domains, if a nuclease domain is deleted or mutated, the Cas protein produced, which is called a nickase, can produce single-strand breaks at the CRISPR RNA (crRNA) recognition sequence in double-stranded DNA, but does not produce double-strand breaks. This nickase can cleave complementary or non-complementary strands, but cannot cleave both at the same time. In certain embodiments, the specificity of targeting double-strand breaks is improved by targeting the nickase to the opposite strands at two locations near the locus. If the nickase cleaves a single strand at two loci, double-strand breaks are formed and can be repaired via HR as described herein. If all nuclease domains of Cas protein (e.g., RuvC and HNH nuclease domains in Cas9 protein; RuvC nuclease domain in Cpf1 protein) are deleted or mutated, the Cas protein produced can have the ability to reduce the two chains of double-stranded DNA cleavage or do not have this ability.

[0179] Also provided herein are variants of the polypeptides disclosed herein. Unless otherwise expressly indicated, polypeptide variants retain their corresponding biological activity. For example, variants of site-directed nuclease polypeptides retain the biological function of the full-length native sequence site-directed nuclease. In another example, variants of non-specific end processing enzymes retain the biological function of the full-length native sequence non-specific end processing enzyme.

[0180] The modification of any polypeptide or protein provided herein is carried out by known methods. By way of example, modification is carried out in the following manner: the nucleotides in the nucleic acid encoding the polypeptide are subjected to site-specific mutagenesis, thereby producing a DNA encoding the modification, and then the DNA is expressed in recombinant cell culture to produce the encoded polypeptide. The technology for carrying out substitution mutations at predetermined sites in DNA with known sequences is well known. For example, M13 primer mutagenesis and PCR-based mutagenesis methods can be used to produce one or more substitution mutations. Any nucleotide sequence provided herein can be codon optimized to change, for example, to maximize expression in a host cell or organism.

[0181] The amino acids in the polypeptides described herein can be any of the 20 naturally occurring amino acids, D-stereoisomers of naturally occurring amino acids, non-natural amino acids, and chemically modified amino acids. Non-natural amino acids (i.e., those that are not naturally found in proteins) are also known in the art, as described, for example, in Zhang et al., "Protein engineering with unnatural amino acids," Curr. Opin. Struct. Biol. 23(4):581-587 (2013); Xie et al., "Adding amino acids to the genetic repertoire," 9(6):548-54 (2005); and all references cited therein. Beta and gamma amino acids are known in the art and are also contemplated herein as non-natural amino acids.

[0182] As used herein, a chemically modified amino acid refers to an amino acid whose side chain has been chemically modified. For example, the side chain can be modified to include a signaling moiety, such as a fluorophore or a radiolabel. The side chain can also be modified to include a new functional group, such as a thiol, a carboxylic acid, or an amino group. Post-translationally modified amino acids are also included in the definition of chemically modified amino acids.

[0183] Conservative amino acid substitutions are also contemplated. By way of example, conservative amino acid substitutions can be made in one or more amino acid residues, for example, in one or more lysine residues of any of the polypeptides provided herein. Those skilled in the art will appreciate that a conservative substitution is the replacement of one amino acid residue with another amino acid residue that is biologically and / or chemically similar. The following eight groups each contain amino acids that are conservative substitutions for one another:

[0184] 1) Alanine (A), glycine (G);

[0185] 2) Aspartic acid (D), glutamic acid (E);

[0186] 3) Asparagine (N), glutamine (Q);

[0187] 4) Arginine (R), Lysine (K);

[0188] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0189] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W);

[0190] 7) Serine (S), Threonine (T); and

[0191] 8) Cysteine ​​(C), methionine (M).

[0192] By way of example, when referring to arginine to serine, conservative substitutions of serine are also contemplated (e.g., threonine). Non-conservative substitutions are also contemplated, such as replacing lysine with asparagine.

[0193] Also provided are DNA constructs comprising a promoter operably linked to a recombinant nucleic acid encoding a fusion protein as described herein or a domain thereof. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. A variety of promoters can be used in the constructs described herein. A promoter is a region or sequence located upstream and / or downstream of the start of transcription and involved in the recognition and binding of RNA polymerase and other proteins to initiate transcription.

[0194] As used herein, the term "promoter" refers to a nucleotide sequence, usually upstream (5') of its coding sequence, which controls the expression of the coding sequence by providing the identification of the RNA polymerase and other factors required for appropriate transcription." Promoter regulatory sequence "is composed of proximal and more distal upstream elements. The promoter regulatory sequence affects the transcription, RNA processing or stability or translation of the relevant coding sequence. Regulatory sequences include enhancers, promoters, non-translated leader sequences, introns and polyadenylation signal sequences. They include natural sequences and synthetic sequences and sequences that may be combinations of synthetic sequences and natural sequences. "Enhancer" is a DNA sequence that can stimulate promoter activity and can be an intrinsic element of the promoter or an inserted heterologous element to enhance the level or tissue specificity of the promoter. It can operate in two orientations (forward or reverse) and can even play a role when moved to the upstream or downstream of the promoter. The meaning of the term "promoter" includes "promoter regulatory sequence".

[0195] The choice of promoter to be included depends on several factors, including but not limited to efficiency, selectability, inducibility, desired expression level, and cell or tissue preferential expression. It is routine for those skilled in the art to regulate the expression of a sequence by appropriately selecting and positioning promoters and other regulatory regions relative to the sequence.

[0196] It has been shown that certain promoters can direct RNA synthesis at a higher rate than others. These are referred to as "strong promoters." It has been shown that certain other promoters direct RNA synthesis at higher levels only in specific types of cells or tissues, and are often referred to as "tissue-specific promoters," or if a promoter preferentially directs RNA synthesis in certain tissues (where RNA synthesis may occur at a reduced level in other tissues), it is referred to as a "tissue-preferred promoter." Because promoters are used to control the expression pattern of one (or more) chimeric genes introduced into plants, there is ongoing interest in isolating novel promoters that can control the expression of one (or more) chimeric genes at certain levels in specific tissue types or at specific plant developmental stages.

[0197] Some promoters can instruct RNA synthesis at relatively similar levels in all tissues of a plant. These are called "constitutive promoters" or "non-tissue-dependent" promoters. Constitutive promoters can be divided into strong, medium, and weak categories based on the effectiveness of their instructing RNA synthesis. Because it is necessary to express one (or more) chimeric genes simultaneously in the different tissues of a plant in many cases to obtain the desired function of one (or more) genes, constitutive promoters are particularly useful in this regard. Although many constitutive promoters have been found and characterized from plants and plant viruses, there is still a continued interest in separating more novel constitutive promoters (synthetic or natural), which can control the expression of one (or more) chimeric genes at different levels and control the expression of multiple genes in the same transgenic plant to carry out gene stacking.

[0198] The recombinant nucleic acid provided herein can be included in an expression cassette for expressing in a target host cell or organism. The cassette will include 5' and 3' regulatory sequences operably connected to the recombinant nucleic acid provided herein for allowing the expression of the fusion protein. The cassette can contain at least one other gene or genetic element to be co-transformed into a cell or organism in addition. In the case of including other genes or elements, these components are operably connected. Alternatively, one or more other genes or elements can be provided on a plurality of expression cassettes. This expression cassette is provided with a plurality of restriction sites and / or recombination sites so that the insertion of the polynucleotide is under the transcriptional regulation of the regulatory region. The expression cassette can contain a selective marker gene in addition. The expression cassette will include in the reverse direction of 5' to 3' transcription: transcription and translation initiation region (i.e., promoter), polynucleotides of the present invention, and transcription and translation termination region (i.e., termination region) that work in the target cell or organism. The promoters of the present invention are capable of directing or driving the expression of a coding sequence (i.e., a nucleic acid sequence that is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, ncRNA, lncRNA, sense RNA, or antisense RNA, regardless of whether the RNA is then translated to produce a protein) in a host cell. The regulatory regions (i.e., the promoter, transcriptional regulatory regions, and translational termination regions) may be endogenous or heterologous to the host cell or to each other. As used herein, "heterologous" with respect to a sequence is a sequence that is derived from a foreign species or, if derived from the same species, has been substantially modified from its native form in composition and / or genomic locus by deliberate human intervention.

[0199] Additional regulatory signals include, but are not limited to, transcription initiation start sites, operators, activators, enhancers, other regulatory elements, ribosome binding sites, initiation codons, termination signals, etc. See Sambrook et al. (1992) Molecular Cloning: A Laboratory Manual, Maniatis et al., eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York); Davis et al., eds. (1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory Press), Cold Spring Harbor, New York, and references cited therein.

[0200] The expression cassette can also comprise a selective marker gene for selecting transformed cells. For example, marker genes include genes that confer antibiotic resistance, such as those that confer hygromycin resistance, ampicillin resistance, gentamicin resistance, neomycin resistance. Other selective markers are known, and any one can be used.

[0201] In preparing the expression cassette, the various DNA fragments may be manipulated to provide a DNA sequence in the proper orientation and, if appropriate, the proper reading frame. To this end, adapters or linkers may be employed to connect the DNA fragments, or other manipulations may be involved to provide convenient restriction sites, remove excess DNA, remove restriction sites, etc. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitution (e.g., transitions and transversions) may be involved.

[0202] Further provided is a vector, which comprises the recombinant nucleic acid or DNA construct set forth herein. It is envisioned that the vector has the necessary functional elements for guiding and regulating the transcription of the inserted nucleic acid. These functional elements include, but are not limited to, a promoter, a region upstream or downstream of the promoter (such as an enhancer that can regulate the transcriptional activity of the promoter), an origin of replication, an appropriate restriction site for promoting the cloning of the inserted sequence adjacent to the promoter, an antibiotic resistance gene or other marker that can be used to select cells containing the vector or the vector containing the inserted sequence, an RNA splicing junction, a transcription termination region, or any other region that can be used to promote the expression of an inserted gene or a hybrid gene. Generally speaking, see Sambrook et al. Molecular Cloning: A Laboratory Manual [Molecular Cloning: Laboratory Manual], 4th edition, Cold Spring Harbor Laboratory Press [Cold Spring Harbor Laboratory Press], Cold Spring Harbor [Cold Spring Harbor], 2012. The vector can be, for example, a plasmid.

[0203] The conversion of cell can be stable or transient.Therefore, transgenic cell of the present invention, vegetable cell, plant and / or plant part can be by stably transformed or transient transformation." conversion " can refer to nucleic acid molecule being transferred in the genome of host cell, produces genetically stable heredity.In certain embodiments, be introduced into plant, plant part and / or vegetable cell and be via bacteria-mediated conversion, particle bombardment conversion, calcium phosphate-mediated conversion, cyclodextrin-mediated conversion, electroporation, liposome-mediated conversion, nanoparticle-mediated conversion, polymer-mediated conversion, virus-mediated nucleic acid delivery, whisker-mediated nucleic acid delivery, microinjection, ultrasonic treatment, infiltration, polyethylene glycol-mediated conversion, protoplast transformation or make nucleic acid be introduced into any other electrical, chemical, physical and / or biological mechanism or its any combination in plant, plant part and / or its cell and carry out.

[0204] The procedures for transforming plants are well known and conventional in the art and are generally described in the literature. Non-limiting examples of methods for plant transformation include transformation via bacteria-mediated nucleic acid delivery (e.g., via bacteria from the genus Agrobacterium), virus-mediated nucleic acid delivery, silicon carbide or nucleic acid whisker-mediated nucleic acid delivery, liposome-mediated nucleic acid delivery, microinjection, microparticle bombardment, calcium phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, nanoparticle-mediated transformation, sonication, infiltration, PEG-mediated nucleic acid uptake, and any other electrical, chemical, physical (mechanical) and / or biological mechanism that allows the introduction of nucleic acids into plant cells, including any combination thereof. General guides to various plant transformation methods known in the art include Miki et al. ("Procedures for Introducing Foreign DNA into Plants," in Methods in Plant Molecular Biology and Biotechnology, Glick, BR and Thompson, JE, eds. (CRC Press, Inc., Boca Raton, 1993), pp. 67-88) and Rakowoczy-Trojanowska (Cell Mol Biol Lett 7:849-858 (2002)).

[0205] Agrobacterium-mediated transformation is a common method for transforming plants due to its high transformation efficiency and due to its wide applicability with many different species. Agrobacterium-mediated transformation typically involves transferring the binary vector carrying the foreign DNA of interest to an appropriate Agrobacterium strain, which may depend on the complement of the vir genes carried by the host Agrobacterium strain on a co-existing Ti plasmid or chromosomally (Uknes et al., 1993, Plant Cell [plant cells] 5: 159-169). Escherichia coli carrying a recombinant binary vector can be used, and an auxiliary Escherichia coli strain (the auxiliary Escherichia coli strain carries a plasmid capable of moving the recombinant binary vector into the target Agrobacterium strain) is passed through a three-parent mating procedure to achieve the transfer of the recombinant binary vector to Agrobacterium. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by nucleic acid transformation ( and Willmitzer 1988, Nucleic Acids Res 16:9877).

[0206] Plant transformation by recombinant Agrobacterium typically involves co-cultivation of Agrobacterium with explants from the plant and follows methods well known in the art. Transformed tissue is typically regenerated on selective media carrying an antibiotic or herbicide resistance marker between the binary plasmid T-DNA borders.

[0207] Another method for transforming plants, plant parts and plant cells involves advancing inert or biologically active particles on plant tissues and cells. See, for example, U.S. Patent Nos. 4,945,050; 5,036,006 and 5,100,792. Generally speaking, this method involves advancing inert or biologically active particles at plant cells under conditions effective to penetrate the outer surface of the cell and provide for incorporation into its interior. When utilizing inert particles, the vector can be introduced into the cell by coating the particles with a vector containing the target nucleic acid. Alternatively, one or more cells can be surrounded by the vector so that the vector is carried into the cell by stimulation of the particle. Biologically active particles (e.g., dried yeast cells, dried bacteria or bacteriophages, each containing one or more nucleic acids intended to be introduced) can also be advanced into plant tissue. As used herein, the phrase "biolistic transformation" refers to a method of introducing RNA or DNA directly into cells (e.g., plant cells) in which the RNA or DNA is mixed with heavy metal particles (e.g., tungsten or gold) and released into the cells (e.g., plant cells) using high-speed pressure to allow the RNA or DNA to penetrate the cells (e.g., penetrate the plant cell wall).

[0208] The CRISPR / Cas system can also be used to edit the genome of a host cell or organism. As described in detail above, the "CRISPR / Cas" system refers to a class of bacterial systems that are widely used to defend against foreign nucleic acids. Any of the CRISPR / Cas system components described herein can be used to introduce fusion proteins, recombinant nucleic acids or systems into the genome of a host cell or organism. Methods for genome editing mediated by the CRISPR / Cas system are known in the art. It should be understood that the use of the CRISPR / Cas system for introducing fusion proteins, recombinant nucleic acids or systems described herein into the genome of a host cell or organism is different from the specific methods and systems provided herein.

[0209] On the other hand, provided herein are systems that can be used to edit one or more nucleic acids. These systems include one or more of the fusion proteins (or recombinant nucleic acids, constructs, vectors or host cells) described above. In certain embodiments, these systems further include one or more other elements that can be used to edit one or more nucleic acids. For example, provided herein are systems that can further include donor polynucleotides. As another example, systems including fusion proteins containing Cas nucleases can further include one or more guide nucleic acids and / or one or more donor polynucleotide sequences.

[0210] In some cases, the systems and methods described herein include at least one guide nucleic acid polynucleotide. In some cases, the systems and methods described herein include multiple guide nucleic acids. In certain embodiments, the polynucleotide can be deoxyribonucleic acid (DNA). In some cases, the DNA sequence can be single-stranded or double-stranded. In certain embodiments, the at least one guide nucleic acid polynucleotide can be ribonucleic acid (guide RNA).

[0211] In certain embodiments, nuclease can be compounded with at least one guide RNA polynucleotide.The at least one guide RNA polynucleotide can include a nucleic acid targeting region, and the nucleic acid targeting region includes a sequence complementary to the nucleic acid sequence on the targeting polynucleotide (such as a targeting genomic locus or a gene) to give the sequence specificity of the nuclease targeting. In certain embodiments, the guide nucleic acid is a single guide nucleic acid including crRNA. In certain embodiments, the guide nucleic acid is a single guide nucleic acid including crRNA but lacking tracrRNA. The crRNA can include a nucleic acid targeting section (e.g., a spacer region) of the guide nucleic acid and a nucleotide segment of half the double-stranded duplex of the Cas protein binding section of the guide nucleic acid that can form a segment.

[0212] In some embodiments, the length of the nucleic acid targeting region (e.g., a spacer) of a guide nucleic acid is 20 nucleotides. In some embodiments, the length of the nucleic acid targeting region of a guide nucleic acid is 19 nucleotides. In some embodiments, the length of the nucleic acid targeting region of a guide nucleic acid is 18 nucleotides. In some embodiments, the length of the nucleic acid targeting region of a guide nucleic acid is 17 nucleotides. In some embodiments, the length of the nucleic acid targeting region of a guide nucleic acid is 16 nucleotides. In some embodiments, the length of the nucleic acid targeting region of a guide nucleic acid is 21 nucleotides. In some embodiments, the length of the nucleic acid targeting region of a guide nucleic acid is 22 nucleotides.

[0213] The length of the nucleotide sequence of the guide nucleic acid that is complementary to the nucleotide sequence of the target nucleic acid (target sequence) can be, for example, at least about 12 nt, at least about 15 nt, at least about 18 nt, at least about 19 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 35 nt, or at least about 40 nt. The length of the nucleotide sequence of the guide nucleic acid that is complementary to the nucleotide sequence of the target nucleic acid (target sequence) can be from about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50 nt, from about 12 nt to about 45 nt, from about 12 nt to about 40 nt, from about 12 nt to about 35 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, from about 12 nt to about 19 nt, from about 19 nt to about 20 ... From about 19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about 60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about 20 nt to about 45 nt, from about 20 nt to about 50 nt, or from about 20 nt to about 60 nt.

[0214] The protospacer sequence of the targeting polynucleotide can be identified by identifying the protospacer adjacent motif (PAM) in the region of interest and selecting a region of desired size upstream or downstream of the PAM as the protospacer. The corresponding spacer sequence can be designed by determining the complementary sequence of the protospacer region.

[0215] Spacer sequences can be identified using a computer program (e.g., machine readable code). The computer program can use variables such as predicted melting temperature, secondary structure formation and predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, GC%, genomic occurrence frequency, methylation status, SNP presence, etc.

[0216] The percent complementarity between a nucleic acid targeting sequence (e.g., a spacer sequence of at least one guide polynucleotide as disclosed herein) and a target nucleic acid (e.g., a protospacer sequence of one or more target loci as disclosed herein) can be at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%. The percent complementarity between a nucleic acid targeting sequence and a target nucleic acid can be at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% over about 20 consecutive nucleotides.

[0217] The guide nucleic acid of the system of the present disclosure can include modifications or sequences that provide additional desired characteristics (e.g., stability of modification or regulation; subcellular targeting; tracking with fluorescent labels; binding sites for proteins or protein complexes, etc.). Examples of such modifications include, for example, a 5' cap (7-methylguanylate cap (m7G)); a 3' polyadenylation tail (3' poly (A) tail); a riboswitch sequence (e.g., to allow regulated stability and / or regulated accessibility of proteins and / or protein complexes); a stability control sequence; a sequence that forms a dsRNA duplex (hairpin); a modification or sequence that targets RNA to a subcellular location (e.g., nucleus, mitochondria, chloroplasts, etc.); a modification or sequence that provides for tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a portion that facilitates fluorescence detection, a sequence that allows fluorescence detection, etc.); a modification or sequence that provides a binding site for a protein (e.g., a protein that acts on DNA, including transcriptional activators, transcriptional repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, and combinations thereof).

[0218] The guide nucleic acid may comprise one or more modifications (e.g., base modifications, backbone modifications) to provide a nucleic acid with new or enhanced characteristics (e.g., improved stability). The guide nucleic acid may comprise a nucleic acid affinity tag. The nucleoside may be a base-sugar combination. The base portion of the nucleotide may be a heterocyclic base. The two most common types of such heterocyclic bases are purines and pyrimidines. The nucleotide may be a nucleoside further comprising a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides comprising pentofuranosyl sugars, the phosphate group may be linked to the 2', 3', or 5' hydroxyl portion of the sugar. When forming the guide nucleic acid, the phosphate group may covalently link adjacent nucleosides to form a linear polymer compound. Furthermore, the corresponding ends of this linear polymer compound may be further linked to form a cyclic compound; however, a linear compound may be suitable. In addition, the linear compound may have internal nucleotide base complementarity and may therefore be folded in a manner that facilitates the production of a fully or partially double-stranded compound. In addition, within the guide nucleic acid, the phosphate group may generally be involved in forming the internucleoside backbone of the guide nucleic acid. The linkage or backbone of the guide nucleic acid can be a 3' to 5' phosphodiester linkage.

[0219] In some embodiments, the at least one guide RNA polynucleotide of the systems or methods provided herein can bind to at least a portion of a genome (e.g., a plant genome) or a gene (e.g., a plant gene). In some cases, the at least one guide RNA polynucleotide is capable of complexing with a site-directed nuclease to direct the site-directed nuclease to target a portion of a target nucleic acid (e.g., a site in a genome or gene).

[0220] In some embodiments, the systems described herein include at least two (e.g., at least three, at least four, at least five, or at least six) different guide RNA polynucleotides capable of forming a complex with a site-directed nuclease. DETAILED DESCRIPTION

[0221] The following description and examples illustrate various aspects and embodiments of the compositions and methods of the present invention. The specific examples are not intended to limit the scope of the compositions and methods. Rather, the examples merely provide non-limiting examples of various compositions and methods that are at least within the scope of the disclosed compositions and methods. The descriptions should be read from the perspective of one of ordinary skill in the art; therefore, they do not necessarily include information that would be familiar to a skilled artisan.

[0222] One aspect of the present invention is a mutant Mb2Cas12a polypeptide comprising at least one amino acid substitution introduced into the wild-type Mb2Cas12a polypeptide sequence of SEQ ID NO: 1. In one embodiment, the mutant Mb2Cas12a polypeptide comprises at least one amino acid substitution occurring at a position selected from the group consisting of: D172, F357, F547, A742, E797, Y819, E913, I914, L917, N918, V921, H939, and Y1172. In another embodiment, the at least one amino acid substitution is selected from the group consisting of: D172R, D172K, F357W, F547Y, A742S, E797A, Y819F, I914K, L917V, N918A, N918K, V921K, V921Q, H939Q and Y1172N. In a further embodiment, the polypeptide comprises a sequence selected from the group consisting of: SEQ ID NO: 3, 5, 26, 35, 57, 58, 64, 76, 80, 85, 86, 87, 88 and 127.

[0223] Another aspect of the present invention is a mutant Mb2Cas12a polypeptide comprising at least one domain swap. In one embodiment, the mutant Mb2Cas12a polypeptide comprises a domain swap occurring at a domain selected from the group consisting of: WED-MR1, WED-MR2, BH, Up-seq, and Nuc. In another embodiment, the WED-MR1 domain is replaced by a sequence selected from the group consisting of: SEQ ID NOs: 24, 29, and 30. In another embodiment, the WED-MR2 domain is replaced by a sequence selected from the group consisting of: SEQ ID NOs: 25, 32, and 33. In yet another embodiment, the BH domain is replaced by a sequence selected from the group consisting of: SEQ ID NOs: 40, 41, and 42. In another embodiment, the Up-seq domain is replaced by a sequence selected from the group consisting of: SEQ ID NOs: 59, 60, 61, 62, and 63. In yet another embodiment, the Nuc domain is replaced by a sequence selected from the group consisting of: SEQ ID NOs: 65, 66, and 67. In further embodiments, the polypeptide comprises a sequence selected from the group comprising SEQ ID NO: 37, 59, 60, 61, 62, 63, 74, 75, 77, and 78.

[0224] Another aspect of the present invention is a mutant Mb2Cas12a polypeptide comprising at least one amino acid substitution and at least one domain swap. In one embodiment, the mutant Mb2Cas12a polypeptide comprises a sequence selected from the group consisting of: SEQ ID NO: 36, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, and 107.

[0225] Another aspect of the present invention is a method for editing a plant genome, the method comprising contacting the plant genome with a mutant Mb2Cas12a polypeptide of the aforementioned embodiment. In an embodiment, the method for editing a plant genome further comprises a guide RNA. In another embodiment, the guide RNA is encoded by a sequence comprising SEQ ID NO: 18-21.

[0226] Another aspect of the present invention is an edited plant obtained by the method of the aforementioned embodiment.

[0227] Another aspect of the present invention is a construct or plasmid comprising a polynucleotide sequence encoding the mutant Mb2Cas12a polypeptide of the aforementioned embodiment. One aspect is a non-human cell comprising a construct or plasmid encoding the mutant Mb2Cas12a polypeptide.

[0228] Examples

[0229] We improved the Mb2Cas12a enzyme activity through protein engineering. Our approach was to rationally design and swap functional domains based on the protein structures of available Cas12a homologs in different states, including MbCas12a-22581 homologs from the same species as Mb2Cas12a (Table 1). The primary amino acid sequence identity of the two homologs was 94.7%, so MbCas12a-22581 served as a good reference for studying the function of Mb2Cas12a, as the latter had no reference structure.

[0230] Table 1: Available Cal2a orthologs and their structures.

[0231]

[0232]

[0233] "PDB" refers to the Protein Data Bank, a public database for archiving protein structural information. See www.rcsb.org.

[0234] In the examples below, all constructs containing coding sequences for engineered or wild-type Mb2Cas12a for maize transformation had the same expression cassette configuration and enzyme configuration ( Figure 1 ). Upstream of the coding sequence is the sugarcane ubiquitin promoter ("prSoUbi4"; SEQ ID NO: 7). The coding sequence of Mb2Cas12a (the sequence depends on the variant used) is fused to the SV40 nuclear localization signal ("NLS") (SEQ ID NO: 8) at the N-terminus via a flexible peptide linker (30 amino acids (GGGGS) 6; SEQ ID NO: 9), and the same peptide linker is fused to two SV40 NLSs at the C-terminus (separated by a short linker (8 amino acids (SGGS) 2; SEQ ID NO: 10). The coding sequence is codon-optimized for maize and linked to the Agrobacterium tumefaciens nopaline synthase gene terminator ("tNOS"; SEQ ID NO: 11). The crRNA array comprising four crRNAs is flanked by ribozyme HH (SEQ ID NO: 12) at the 5' end of the array and HDV (SEQ ID NO: 13) at the 3' end of the array, and is controlled by another set of regulatory components that are identical to those controlling the expression of the selected Mb2Cas12a sequence. The crRNA array expresses four crRNAs targeting four different maize genes: Waxy1 (ZmWx1), UDP-glucosyltransferase benzoxazinoid 9 (ZmBX9), Glossy2 (ZmGL2), and BCL2-related X protein (ZmBINa). The natural Mb2Cas12a mature direct repeat (DR) is used as the crRNA scaffold for design. The crRNA array is represented by SEQ ID NO: 14 and comprises ribozymes.

[0235] The construct was stably transformed into immature maize embryos using a standard transformation protocol (Zhong et al., 2018). Leaf sheath tissue of regenerated plantlets was sampled for DNA extraction, and transgenic plants were identified by TaqMan qPCR assay. Each of the four target sites was confirmed using sequencing and analyzed by TaqMan qPCR assay to determine the editing efficiency of each target site.

[0236] 1. The D172 variant of Mb2Cas12a improves editing efficiency.

[0237] To determine whether the Mb2Cas12a D172R or D172K mutation would improve enzyme activity in maize, two constructs were constructed to express each variant and then transformed into maize for event analysis.

[0238] Table 2 summarizes the SDN1 efficiency at the four target sites. Compared to the control (wild-type Mb2Cas12 (SEQ ID NO: 1), the D172R variant (SEQ ID NO: 3) significantly improved the SDN1 efficiency, except for the first crRNA in the array, which had a slightly reduced efficiency. Compared to the control, the D172K variant (SEQ ID NO: 5) significantly improved the SDN1 efficiency of all target sites, although the increase in the last three crRNAs was less than that of the D172R variant.

[0239] Table 2: SDN1 editing efficiency of Mb2Cas12a variants of D172 in maize.

[0240]

[0241] Efficiency was measured as the percentage of the number of plants with insertion / deletion (indel) mutations divided by the total number of transgenic plants.

[0242] 2. Mutation variants at the WED-III domain improved the editing efficiency of Mb2Cas12a.

[0243] By processing the 5' end of each crRNA in the crRNA array to release a single functional crRNA, Cas12a has an intrinsic gRNA self-processing ability. In order to improve the gRNA processing ability of Mb2Cas12a, we identified the conserved hydrophilic amino acid residue, glutamic acid, in the species Cas12a of Moraxella bovis at position 797 (E797) of Mb2Cas12a based on comparative analysis. In other Cas12a orthologs (such as Lb, As and Fn), the corresponding position is a hydrophobic amino acid, i.e., L807 in AsCas12a, A766 in LbCas12a, and A850 in FnCas12a. We assume that the hydrophilic side chain of E797 may affect the local spatial structure due to interactions with surrounding atoms. Therefore, we evaluated the relationship between the E797A mutation of Mb2Cas12a and D172R in stable transformation. At the same time, a microregion (MR1) with lower acidic / basic amino acid content was identified in Moraxella bovis species compared to Lb, As and FnCas12a (named WED-MR1; SEQ ID NO: 22) (Table 3). Another microregion (MR2) with high diversity in Cas12a orthologs (Table 4) was found (named WED-MR2; SEQ ID NO: 23). We performed microregion exchange by replacing WED-MR1 with the homolog LKKEELVV (SEQ ID NO: 24) of LbCas12a in the D172R form of Mb2Cas12a. We further replaced WED-MR2 with the homolog TTTLS (SEQ ID NO: 25) of LbCas12a in the wild-type form of Mb2Cas12a.

[0244] Table 3. Comparison of amino acids of WED-MR1 among Cas12a orthologs.

[0245]

[0246] Table 4. Comparison of amino acids of WED-MR2 among Cas12a orthologs.

[0247]

[0248]

[0249] As summarized in Table 5, the SDN1 efficiency at four target sites is compared with wild-type Mb2Cas12a (SEQ ID NO: 1). Mb2Cas12a variants (SEQ ID NO: 26) with single amino acid mutation E797A significantly improve editing efficiency at four target sites. Meanwhile, the mutation is also shown in a similar contribution under D172R background (i.e., D172R and E797A; SEQ ID NO: 35), which further confirms the importance of the amino acid for Mb2Cas12a enzymatic activity. For encoding Mb2Cas12a variants (i.e., SEQ ID NO: 36, exchanged with the homolog of LbCas12a) comprising D172R and WED-MR1, the efficiency of the first two crRNAs is enhanced, although the efficiency of the last crRNA is observed to be slightly reduced. Compared to 26411, the efficiency at the first two gRNAs of 27493 (i.e., SEQ ID NO: 37, exchanged with a homolog of LbCas12a) carrying WED-MR2-Lb was improved.

[0250] Table 5. SDN1 editing efficiency of Mb2Cas12a WED-III mutant variants in maize

[0251]

[0252] Efficiency was measured as the percentage of the number of plants with insertion / deletion (indel) mutations divided by the total number of transgenic plants.

[0253] 3. BH domain swap variants or extended BH domain significantly improved Mb2Cas12a activity.

[0254] The bridge helix ("BH") domain is a central helix in the protein that structurally connects the REC and Nuc lobes, which can affect cutting accuracy and trimming activity, increase the specificity of Cas12a, and enable the apo enzyme to adopt a closed state, thereby promoting efficient crRNA loading. Destroying the α-helical properties of BH in Cas12a changes the trimming activity and cleavage rate (Worle et al., 2021). The BH domains in Cas12a orthologs were compared (Table 6). The comparison shows that the α-helix length of the BH domain in MbCas12a-22581 is shorter than that of other orthologs, and therefore may affect enzyme activity.

[0255] Table 6. Comparison of BH domains among Cas12a orthologs.

[0256]

[0257] Based on this analysis, we created a series of Mb2Cas12a variants with high enzymatic activity that carry microdomain domain exchanges from other orthologs, as listed in Table 8. We also changed amino acids by rational design to extend the length of the α helix in the BH domain and made two variants (L917V+N918K and I914K+L917V+N918A in Table 8). At the same time, we identified a high diversity region ("Up-seq") located upstream of the BH domain (Table 7). We also identified the hydrophobic amino acid Val921, which has different properties compared to the hydrophilic amino acids AsCas12a (Gln956), LbCas12a (Gln888) or FnCas12a (Lys969) at the same position in the following. The main chain carbonyl group of Gln956 in the bridge helix can form a hydrogen bond with the side chain of Lys468 in the REC2 domain of AsCas12a (Yamano et al., 2016). At the same time, the amide group of the side chain forms an interaction with the sugar-phosphate backbone of the guide RNA through hydrogen bonds. The homolog Val931 in MbCas12a-22581 cannot form this interaction because the distance between Val931 and the sugar-phosphate backbone exceeds Based on the above analysis and the high consistency between Mb2Cas12a and MbCas12a-22581, we created the single amino acid mutations listed in Table 8.

[0258] Table 7. Comparison of Up-seq regions among Cas12a orthologs.

[0259]

[0260] The SDN1 efficiency at the four target sites was compared as summarized in Table 8. All of these variants significantly improved the SDN1 efficiency at the four target sites compared to the control (26411). Construct 26445 carrying variant BH2-As showed the best performance in editing the four target sites in maize.

[0261] Table 8. SDN1 editing efficiency of Mb2Cas12a variants in the BH domain in maize.

[0262]

[0263] 4. Variants of the Nuc domain improved Mb2Cas12a enzyme activity.

[0264] The endonuclease active site of FnCas12a is located at the interface of the RuvC and Nuc domains, responsible for the sequential cleavage of target and non-target DNA chains (Swarts et al., 2017). Conserved polar residues Arg1226 and Asp1235 and partially conserved Ser1228 are clustered near the active site of the RuvC domain in AsCas12a. These residues are key residues for endonuclease activity in the Nuc domain (Yamano et al., 2016). Peptide alignment of Cas12a orthologs showed that the residues around this region are very diverse (Table 9).

[0265] Table 9. Comparison of microregions of the Nuc domain in Cas12a orthologs.

[0266]

[0267] These key residues are located in a small area, and we replaced this small area with the homologous sequences of LbCas12a and AsCas12a, respectively (Table 10). The point mutation Y1172N in this area was also evaluated (Table 10). At the same time, considering that the entire Nuc domain has little interaction with other domains, according to our structural analysis, we also replaced the entire Nuc domain with the homologous sequences of LbCas12a and AsCas12a, respectively (Table 10).

[0268] Table 10. SDN1 editing efficiency of Mb2Cas12a variants in the Nuc domain in maize.

[0269]

[0270] As shown in the editing efficiencies summarized in Table 10, three variants, variant Nuc1-As (in construct 26441) containing the homologous Nuc domain of AsCas12a, variant Y1172N (in construct 26438) containing a single amino acid mutation, and variant Nuc2-Lb (in construct 26553) containing the entire Nuc domain replacement of LbCas12a, significantly improved editing efficiency.

[0271] 5. The Mb2Cas12a F357W variant improves editing efficiency.

[0272] For the Cas12a system, even though our crRNA design uses a 23-nucleotide spacer length, the interaction between the target DNA sequence and the crRNA only forms 20 base pairs. This is partly because Trp382 interacts with the 20th base pair of DNA and crRNA, resulting in the destruction of base pairs at position 21 and beyond. Therefore, this amino acid residue is crucial for the formation of the Cas12a-DNA-crRNA triplex structure.

[0273] In AsCas12a, Trp382 is paired with C20:dG20 in a heteroduplex form to form a stacking interaction (i.e., "dG20" refers to the base at position 20 on the DNA target strand; "C20" refers to the base at position 20 in the crRNA space), thereby preventing base pairing between A21 and dT21. In addition, the W382A mutation of AsCas12a reduces enzyme activity (Yamano et al., 2016), which means that the W382 residue contributes to enzyme performance. Sequence alignment shows that in most Cas12a orthologs, there are functionally conserved aromatic amino acids at this position (Table 11).

[0274] Table 11. Alignment analysis of Phe357 of Mb2Cas12a.

[0275] Amino acid comparison LbCas12a W355 AsCas12a W382 FnCas12a Y410 MbCas12a-22581 F370 Mb2Cas12a F357

[0276] When the side chain was further analyzed, the homologous Phe357 of Mb2Cas12a was different from the conserved Trp or Tyr residues, which have hydrogen donor or acceptor atoms. Since phenylalanine has no hydrogen donor or acceptor in its side chain, this indicates that there is little interaction between Phe357 of Mb2Cas12a and the RNA-DNA heteroduplex, further suggesting that Phe357 may reduce enzyme activity. To test this hypothesis, a single residue mutation F357W was constructed to evaluate its function in maize.

[0277] The SDN1 efficiency at the four target sites was compared as summarized in Table 12. The F357W variant (26446) significantly improved the SDN1 efficiency at the four target sites compared to the control (26411).

[0278] Table 12. SDN1 editing efficiency of the F357W variant in the REC2 domain in maize.

[0279]

[0280] 6. Enhancing the interaction between Mb2Cas12a and the 5' direct repeat of crRNA improved SDN1 efficiency.

[0281] In order to crack the target DNA sequence, Cas12a protein is connected to the crRNA molecule in the form of a ribonucleoprotein (" RNP ") complex. Stronger Cas12a-crRNA interaction, i.e., more stable Cas12a-crRNA RNP complex, can probably improve DNA binding efficiency and crRNA-dependent DNA enzyme activity. Studies on various Cas12a-crRNA complexes have shown that Cas12a is combined with the pseudoknot structure formed by 5' direct repeats (" DR ") in mature crRNA. We compared the Cas12a-crRNA binary complex structure of LbCas12a with that of MbCas12a-22581. We identified four amino acid residues (see Table 13), which differ in their performance between LbCas12a and MbCas12a-22581 in terms of interaction with DR.

[0282] Table 13. Amino acid residues linked to the pseudoknot.

[0283] LbCas12a MbCas12a-22581 Mb2Cas12a Y516 F557 F547 S713 A752 A742 F789 Y829 Y819 Q906 H949 H939

[0284] Y516 and S713 of LbCas12a interact with the ribose phosphate backbone of DR, while the corresponding residues in MbCas12a-22581 (F557 and A752, respectively) cannot form hydrogen bonds due to the side chain properties and do not interact with DR. Q906 of LbCas12a and its counterpart (H949) in MbCas12a-22581 may interact with the nucleobase in DR. But they tend to interact with different nucleobases, and the interaction between H949 of MbCas12a-22581 and U7 of DR (i.e., uracil at position 7 of the direct repeat) is more likely to interfere with the base pairing in the pseudoknot structure between U7 and A17 (i.e., adenine at position 17 of the direct repeat). F789 of LbCas12a does not interact with DR, whereas the counterpart (Y829) in MbCas12a-22581 has a strong potential to form hydrogen bonds with DR due to the additional hydroxyl group in the side chain.

[0285] Based on these analyses, we prepared a series of single mutation Mb2Cas12a variants (Table 14) by replacing each of the four residues in Mb2Cas12a with the corresponding residues in LbCas12a. Similar to the previous example, a nuclear localization signal and a polypeptide linker were added to each variant, and a binary vector was constructed to produce transgenic maize plants and target four maize genes. Except for the H939Q variant in 27501, the other variants significantly improved the editing efficiency at the four target sites.

[0286] Table 14. Comparison of SDN1 editing efficiency in maize.

[0287]

[0288]

[0289] 7. A combination of single region changes improved SDN1 efficiency at four target sites in maize.

[0290] To further evaluate the synergistic effect of mutations or small peptide substitutions validated in previous experiments on improving enzyme activity, a series of constructs combining various mutations were prepared as shown in Table 15. Compared to the control (26411), all combinatorial variants tested improved efficiency at all target sites, with the exception of one variant in 27381, which had a slight decrease in efficiency at the first target site.

[0291] Table 15. Comparison of SDN1 editing efficiency of combinatorial variants and wild type in maize.

[0292]

[0293]

[0294] Example 8. Construct annotation. 26411 Annotation:

[0295]

[0296]

[0297]

[0298] 26363 Notes:

[0299]

[0300]

[0301] 26410 Notes:

[0302]

[0303]

[0304] 27731 Notes:

[0305]

[0306]

[0307] 26840 Notes:

[0308]

[0309]

[0310] 26841 Notes:

[0311]

[0312]

[0313] 27493 Notes:

[0314]

[0315]

[0316] 26442 Notes:

[0317]

[0318]

[0319]

[0320] 26443 Notes:

[0321]

[0322]

[0323] 26623 Notes:

[0324]

[0325]

[0326] 26444 Notes:

[0327]

[0328]

[0329] 27031 Notes:

[0330]

[0331]

[0332] 26445 Notes:

[0333]

[0334]

[0335] 27030 Notes:

[0336]

[0337]

[0338] 27927 Notes:

[0339]

[0340]

[0341]

[0342] 26440 Notes:

[0343]

[0344]

[0345] 26441 Notes:

[0346]

[0347]

[0348] 26438 Notes:

[0349]

[0350]

[0351]

[0352] 26553 Notes:

[0353]

[0354]

[0355] 27215 Notes:

[0356]

[0357]

[0358] 26446 Notes:

[0359]

[0360]

[0361]

[0362] 27495 Notes:

[0363]

[0364]

[0365] 27745 Notes:

[0366]

[0367]

[0368] 27747 Notes:

[0369]

[0370]

[0371] 27501 Notes:

[0372]

[0373]

[0374] 27218 Notes:

[0375]

[0376]

[0377] 27025 Notes:

[0378]

[0379]

[0380] 27216 Notes:

[0381]

[0382]

[0383] 27219 Notes:

[0384]

[0385]

[0386] 27220 Notes:

[0387]

[0388]

[0389]

[0390] 27225 Notes:

[0391]

[0392]

[0393] 27228 Notes:

[0394]

[0395]

[0396] 27223 Notes:

[0397]

[0398]

[0399]

[0400] 27224 Notes:

[0401]

[0402]

[0403] 27310 Notes:

[0404]

[0405]

[0406] 27311 Notes:

[0407]

[0408]

[0409] 27320 Notes:

[0410]

[0411]

[0412] 27321 Notes:

[0413]

[0414]

[0415]

[0416] 27322 Notes:

[0417]

[0418]

[0419] 27381 Notes:

[0420]

[0421]

[0422] 27382 Notes:

[0423]

[0424]

[0425]

[0426] 27383 Notes:

[0427]

[0428]

[0429] 27325 Notes:

[0430]

[0431]

[0432] 27323 Notes:

[0433]

[0434]

[0435]

[0436] 27926 Notes:

[0437]

[0438]

Claims

1. A mutant Mb2Cas12a polypeptide comprising at least one amino acid substitution introduced into the wild-type Mb2Cas12a polypeptide sequence of SEQ ID NO:

1.

2. The mutant Mb2Cas12a polypeptide of claim 1, wherein the at least one amino acid substitution occurs at a position selected from the group consisting of: D172, F357, F547, A742, E797, Y819, E913, I914, L917, N918, V921, H939, and Y1172.

3. The mutant Mb2Cas12a polypeptide of claim 2, wherein the at least one amino acid substitution is selected from the group consisting of D172R, D172K, F357W, F547Y, A742S, E797A, Y819F, I914K, L917V, N918A, N918K, V921K, V921Q, H939Q, and Y1172N.

4. The mutant Mb2Cas12a polypeptide of claim 3, wherein the polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 3, 5, 26, 35, 57, 58, 64, 76, 80, 85, 86, 87, 88, and 127.

5. A mutant Mb2Cas12a polypeptide comprising at least one domain exchange.

6. mutant Mb2Cas12a polypeptide as claimed in claim 5, wherein the domain exchange occurs at a domain selected from the group consisting of: WED-MR1, WED-MR2, BH, Up-seq and Nuc.

7. mutant Mb2Cas12a polypeptide as claimed in claim 6, wherein the WED-MR1 domain is replaced by a sequence selected from the group consisting of: SEQ ID NO: 24, 29 and 30.

8. mutant Mb2Cas12a polypeptide as claimed in claim 6, wherein the WED-MR2 domain is replaced by a sequence selected from the group consisting of: SEQ ID NO: 25, 32 and 33.

9. mutant Mb2Cas12a polypeptide as claimed in claim 6, wherein the BH domain is replaced by a sequence selected from the group consisting of: SEQ ID NO:40, 41 and 42.

10. The mutant Mb2Cas12a polypeptide of claim 6, wherein the Up-seq domain is replaced by a sequence selected from the group consisting of SEQ ID NOs: 59, 60, 61, 62, and 63.

11. mutant Mb2Cas12a polypeptide as claimed in claim 6, wherein the Nuc domain is replaced by a sequence selected from the group consisting of: SEQ ID NO:65, 66 and 67.

12. The mutant Mb2Cas12a polypeptide of claim 5, wherein the polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 37, 59, 60, 61, 62, 63, 74, 75, 77, and 78.

13. A mutant Mb2Cas12a polypeptide comprising at least one amino acid substitution and at least one domain exchange.

14. The mutant Mb2Cas12a polypeptide of claim 13, wherein the Mb2Cas12a polypeptide sequence is selected from the group consisting of SEQ ID NO: 36, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, and 107.

15. A method of editing a plant genome, comprising contacting the plant genome with a mutant Mb2Cas12a polypeptide as described in claims 1-14.

16. The method of claim 15, further comprising a guide RNA.

17. The method of claim 16, wherein the guide RNA is encoded by a sequence comprising SEQ ID NOs: 18-21.

18. An edited plant obtained by the method of claims 15-17.

19. A construct or plasmid comprising a polynucleotide sequence encoding a mutant Mb2Cas12a polypeptide as described in claims 1-14.

20. A non-human cell comprising the construct or plasmid of claim 19.

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